This grant proposal will investigate the role of cell-surface central nervous system (CNS)-specific heparan- sulfate and chondroitin-sulfate proteoglycans (HSPGs and CSPGs respectively) in human immunodeficiency virus type I (HIV-1) neuropathogenesis. The working hypothesis for these studies is that distinct gene- families of cell-associated proteoglycans play a major role in viral capture and transfer via the brain microvascular endothelial cells (MVECs), as we have demonstrated, and other independent investigators have confirmed. In addition, distinct CNS-specific HSPGs may be required for interactions of viral proteins (HIV-1 Tat) with the blood-brain barrier (BBB) and CNS-derived cells. SPECIFIC AIM 1 will investigate the molecular mechanisms by which HIV-1 interacts with MVEC-specific cell-associated proteoglycans to cross the BBB and infect CMS. This will be accomplished by: 1). Complete characterization of MVEC-specific cell- associated proteoglycans in our experimental cellular systems in vitro, 2). Analysis of the role of MVEC- specific proteoglycans in HIV-1 entry and infection of the BBB and CNS by means of alternative approaches, such as chemical, enzymatic, monoclonal antibodies, short interfering RNA (siRNA), and anti-sense. 3). Investigating the role of MVEC-specific proteoglycans in HIV-1 infection of CNS by both free-virus and cell- associated (transmigration of HIV-1 infected monocytes/T-cells through the BBB) virus in our in vitro human BBB model. SPECIFIC AIM 2 will study the molecular mechanisms by which HIV-1-specific proteins (HIV-1 Tat) interact with CNS-specific cell-associated HSPGs to enter and affect CNS-derived cells. By employing similar approaches as in specific AIM1 (i.e. enzymatic, monoclonal antibodies, RNAi, and anti-sense oligonucleotides) we will dissect the participation of specific gene-families of HSPGs (syndecans, glypicans, and/or others) in HIV-1 Tat uptake by human brain MVECs, neurons and astrocytes. In addition, as a model in our studies, we will also employ the human K562-cell line, which lacks HSPGs and CSPGs, and will be transfected with different constructs expressing various human proteoglycans, that are available in our labs. Results from these studies should improve our understanding of the role of CNS-specific proteoglycans in HIV-1 entry and infection of the brain, and may provide insights into strategies for controlling HIV-1 neuroinvasion and associated neurodegeneration in infected patients.